The use of antibodies labeled with radioactive iodine isotopes has been proposed to detect tumor-associated antigens. For instance, in the U.S. Pat. No. 3,927,193 labeled goat anti-human carcino-embryonic antigen antibodies have been injected to Syrian hamsters inoculated with human carcinoma and shown to localize preferentially in the tumor. It was thus suggested that labeled antibodies might be used to visualize tumors after injection to patients using detectors available in the art. Such diagnostic applications are commonly refered to as immunodiagnostic. As early as 1956, Beierwaltes and coworkers cured a patient with advanced malignant melanoma by injecting large amounts of .sup.131 I-labeled gamma-globulins from a rabbit immunized with the patients' own tumor cells. Further use of directly labeled polyclonal antibodies has not met with equal success. For convenience, this, and other related applications of antibodies, will be refered to as immunotherapy.
Since the discovery of monoclonal antibodies (Kohler and Milstein), monoclonal antibodies capable of specific binding to cells of a particular type, or, in a less specific way, to cells of a few different types, have been obtained in many laboratories and industries. Such monoclonal antibodies are most attractive because they are homogeneous and potentially more specific than polyclonal antibodies extracted from antisera. They have been widely used to identify cells in tissue sections and various biological samples, and to diagnose cancer and metastases in vitro (Gatter et al.). An obvious application of these reagents was to label them with a suitable radioactive isotope and inject them in animals or human in order to visualize in vivo specific cell subsets (e.g. tumors or metastases) using existing devices such as the gamma camera. Another application was to inject large quantities of monoclonal antibodies labeled with radioactive isotopes capable of killing the cells (e.g. malignant cells) to which the antibody became bound.
Isotopes generally used in radioimmunoscintigraphy are: .sup.131 iodine and .sup.123 iodine (covalently coupled to tyrosines of the antibody, Hunter and Greenwood); .sup.111 indium, .sup.99m Tc and other metals (attached directly or by means of suitable chelating agents covalently coupled to the antibody, Hnatowich et al.). For radioimmunotherapy, high linear energy transfer (LET) isotopes are usually preferred (e.g. .sup.131 I, .sup.211 At, .sup.212 Bi).
The state of the art and the major limitations of radioimmunoscintigraphy and radioimmunotherapy have been discussed by Bradwell et al. The essential parameters in these techniques are the fraction of the injected dose specifically localized at the site(s) where target cells are present and the uptake ratio (i.e. the ratio of the concentration of specifically bound antibody to that of the radioactivity present in surrounding normal tissues). These parameters are related in a non-trivial way. Usually the fraction of injected dose localized in the tumor is not much better than 0.1%, and contrast not better than 2 to 3. These figures translate in the common observation that tumors (or other tissue injuries) smaller than 1 to 2 cm in diameter cannot be detected, and that radioimmunotherapy has met with little success so far. Non specific uptake by non-target organs such as the liver, kidneys or bone-marrow is another major limitation of the technique, especially for radioimmunotherapy, where irradiation of the bone marrow often causes the dose-limiting toxicity.
Recently, the use of low molecular weight tracers, such as indium chelates, associated with dual specificity antibody conjugates combining antibodies (or fragments) to the target cells with antibodies (or fragments) to the indium chelate, has been proposed (Reardan et al.). Increased uptake ratios and faster localization of the tracer are expected, since the radioactivity would be associated to low molecular weight structures capable of fast distribution through the body tissues and of rapid clearance. If the radioactive isotope has a rapid radioactive decay, such as .sup.123 I or .sup.99m Tc, images recorded sooner after injection will be obtained with higher activities remaining than with the conventional techniques. Similarly, fast localization and rapid clearance of excess radioactive isotopes, or drugs or toxins would reduce damage to normal cells and tissues in immunotherapy.
However, the tracer may be effectively trapped by excess circulating dual specificity conjugate, and its specific localization and its clearance would be impaired. This is a major limitation of the proposed two-step technique in immunodiagnostic and immunotherapy. To take advantage of the theoretical potential of the method, excess dual specificity conjugate should be removed from the circulation prior to injection of the tracer (Goodwin). This would involve cumbersome in vivo manipulations, which have not been substantiated yet. Thus further improvements of the method are still required.
Other useful techniques for the diagnosis of cancer and tissue injuries which do not necessarily involve the use of antibodies are known to the art. In addition to the techniques derived from X-ray radiography, of which an elaborated version is the computer assisted tomography (CAT scanning), sophisticated detectors have been developed to monitor the magnetic resonance properties of living organisms, and particularly to produce images of organs or whole bodies in a technique called Magnetic Resonance Imaging (MRI). The association of the exquisite spatial resolution of MRI and the specificity of immunological reagents such as the monoclonal antibodies has been contemplated (Unger et al., Curtet et al.). It has been proposed to label monoclonal antibodies to specific cellular antigens with chemical groups capable of enhancing the relaxation of the protons contained in body tissues and fluids, and particularly to use paramagnetic metal ions such as Fe, Mn, or Gd. However, the technique is far from achieving clinically useful results, particularly because the concentrations of relaxation agents that must be deposited at the target sites are very high. Theoretically, the problem would be solved by conjugating several thousand relaxation agents per antibody molecule, but this has not been possible yet without compromising the ability of the antibody to recognize the antigen. Thus, in this area also, substantial advances must occur.
In an entirely different domain of the prior art, a few natural multivalent ligands are recognized to bind more tightly to multivalent receptors than the corresponding monovalent ligands (e.g. binding of IgE and IgM to cell membrane receptors, binding of agregated IgG to the polymeric Fc receptor, or C1q binding to immune complexes). Similarly, synthetic multivalent ligands for receptors such as the DNA have been described (Le Pecq et al.) with increased affinity as compared to the monovalent ligand. However, no useful application of this knowledge has been proposed in the fields of in vivo immunodiagnostic or immunotherapy.